The ability to diagnose cancer at an early stage will greatly enhance chances of treatment success and reduce the mortality and morbidity. Currently there is no molecular test that can diagnose or screen for oral cancer non-invasively. We were able to identify a 7-gene signature that can predict the presence of an oral cancer with an accuracy of 82%. Since 2004, the characterization and diagnostic use of human salivary RNA has been actively pursued in our laboratory. Our proposal, "Collection, Stabilization and Storage of Saliva Samples for Cancer Research," responds to the RFA titled "Innovations in Cancer Sample Preparation" (RFA CA-07-037). We will develop optimal and standardized collection, RNA stabilization, storage and processing protocols to perform gene expression analysis of salivary mRNA. We propose three Specific Aims to address the multifaceted nature of this RFA. Aim 1 will establish optimal extraction methods for mRNA from saliva. In Aim 2 we will determine the intra-individual fluctuation of mRNA levels, define adequate endogenous transcripts for normalization and validate our oral cancer markers and additional new candidates in an independent cohort. In Aim 3 we will determine stability of saliva and expression patterns over periods of up to 6 months with different storage conditions and with stabilizing reagent. In all aims we will apply a new method for the multiplex reverse transcription and pre-amplification with subsequent quantitative PCR of a variety of mRNA transcripts endogenous to saliva. In addition, our set-up allows the implementation of several spike sequences that will yield important insights in the extraction and storage and are ideal controls for clinical application. We are confident that our research can be translated directly into a study for the large validation of oral cancer markers and spark the initiation of a multitude of projects and sample collections for the use of saliva as a diagnostic tool for oral and systemic diseases. Our studies will contribute to the advancement of human saliva as a clinically important body fluid for molecular diagnosis to improve cancer-directed health care.